Organ transplantation is now common for patients with end-stage renal, cardiac, hepatic or pulmonary failure. Currently, allografts of kidney, heart, lung, liver, bone marrow, pancreas (islet cells), cornea, small intestine and skin are routinely performed. Additionally xenografts are now performed using porcine heart valves. Organ transplants, however, evoke a variety of immune responses in the host resulting in graft rejection and Graft-Versus-Host Disease (hereinafter, referred to as "GVHD").
The immune response is primarily triggered by T cells through recognition of alloantigens, and the major targets in transplant rejection are non-self allelic forms of class I and class II Major Histocompatibility Complex (MHC) antigens. In acute rejection, donor's antigen-presenting cells such as dendritic cells and monocytes migrate from the allograft to the regional lymph nodes, where they are recognized as foreign by the recipient's CD4.sup.+ T.sub.H cells, stimulating T.sub.H cell proliferation. Following T.sub.H cells proliferation, a population of effector cells (including cytotoxic CD8.sup.+ T cells and CD4+ T cells) is generated, which migrates and infiltrates to the graft and mediates graft rejection (Noelle et al., FASEB 5(13):2770, 1991).
Whereas acute rejection is a T cell-dependent process, a broad array of effector mechanisms participates in graft destruction. Through the release of cytokines and cell-to-cell interactions, a diverse assembly of lymphocytes including CD4.sup.+ T cells, CD8.sup.+ cytotoxic T cells, antibody-forming B cells and other proinflammatory leukocytes, is recruited into the anti-allograft response. Antigen-presenting graft cells are destroyed directly by cytotoxic CD8.sup.+ T cells. Activated CD4.sup.+ T cells produce interleukin-2 (hereinafter, referred to as "IL-2"), which is essential to the activation of both CD8.sup.+ T cells and B cells. Additionally, CD4.sup.+ T cells produce other cytokines such as IFN-.gamma. and IL-4 that also contribute to the destruction of allograft. Furthermore, interferon-.gamma. (hereinafter, referred to as "IFN-.gamma.") induces increased expression of class I and class II MHC molecules on graft tissue, which is more readily attacked by alloreactive effector cells. IFN-.gamma. enhances macrophage activity and affects many inflammatory cells leading to delayed-type-hypersensitivity reaction and inflammation causing nonspecific damage to the graft. These reactions appear to be the primary cause of the early acute rejection that may occur within the first few weeks after transplant. If untreated, acute rejection progresses to a rapid and severe process that causes destruction of the transplant within a few days.
In the case of bone marrow transplantation, in particular, GVHD (graft-versus-host disease) is another obstacle to survival of transplanted patient& GVHD is a disease in which transplanted marrow cells attack the recipient's cells (Storb, "Pathophysiology and prevention of graft-versus-host disease." In Advances in Immunobiology: Blood cell antigens and bone marrow transplantation, McCullogh and Sandler, editors, Alan, Inc., N.Y., p.337, 1984). A large proportion of GVHD-afflicted individuals dies as a result of GVHD (Weiden et al., "Graft-versus-host disease in allogeneic marrow transplantation", in Biology of Bone-Marrow Transplantation, Gale and Fox, editors, Academic Press, N.Y., p37, 1980).
To protect patients from such fatal damages, various immunosuppressive agents have been employed. Currently, allograft rejection is controlled using immunosuppressive agents such as cydosporin A, azathioprine, corticosteroids including prednisone, and methylprednisolone) cyclophosphamide. Cyclosporin A, the most powerful and most frequently used immunosuppressant, revolutionized the field of organ transplant surgery. Other immunosuppressive agents such as FK506, rapamycin, mycophenolic acid, 15-deoxyspergualin, mimoribine, misoprostol, OKT3 and anti-IL-2 receptor antibodies, have been used in the treatment and/or prevention of organ transplantation rejection (Briggs, Immunology letters, 29(1-2), 89-94, 1991; FASEB 3:3411, 1989). Although the development of new immunosuppressive drugs has led to substantial improvement in the survival of patients, these drugs are associated with a high incidence of side effects such as nephrotoxicity and/or hepatotoxicity.
Because immune responses in transplantation rejection are primarily dependent on T cell, agents that suppress the functions of T cells are likely to be more efficacious drugs. Cyclosporin A (CsA) and FK506 inhibit T cell activation by inhibiting the transcription of cytokine genes. Their action mechanism can be summarized as follows.
Cyclosporin A and FK506 bind to immunophilins such as Cyclophilins and FK506-binding proteins (FKBPs). The drug-immunophilin complexes bind to and inhibit the enzymatic activity of the Calcium- and Calmodulin-dependent serine/threonine phosphatase, Calcineurin. The inactivation of this enzyme blocks the nuclear translocation of the cytoplasmic component of the transcription factor NF-AT Nuclear factor of activating T cells) required for the expression of cytokine genes including IL-2.
However, Cyclosporin A has associated toxicities and side effects when used even at therapeutic doses. Although FK506 is about 10 to 100 times more potent than Cyclosporin A in inhibiting activation-induced IL-2 transcription in vitro and graft rejection in vivo, it also shows the side effect such as neurotoxicity and nephrotoxicity.
Rapamyycin is another potent immunosuppressant that inhibits IL-2-dependent lymphocyte proliferation. The cellular target of rapamycin-FKBP complex, mTOR has a phosphoinositide kinase activity and the mTOR signaling is related to cell cycle progression in response to cytokines.
All these immunosuppressive drugs described above affect the T cell functions; whereas Cyclosporin A and FK506 block the step of T cell activation, rapamycin inhibits the T cell proliferation. However, the cellular targets of these agents are expressed ubiquitously. Compounds that selectively inhibit the target molecules which is specifically expressed in immune cells, especially T cells, will undoubtedly be an ideal and more efficient immunosuppressive drug, Thus, there presently exists a need for improved immunosuppressive agents acting specifically on immune cells.
Inhibition of T cell activation and effector functions is an attractive target for the development of drugs to treat T cell-mediated immunopathologies, like autoimmune diseases as well as transplant rejection. The antigen-specific T cell activation is initiated by T cell receptor (hereinafter, referred to as "TCR")-mediated signal transduction in which many tyrosine kinases or phosphoproteins are involved. Progression of activated T cells into cell proliferation is subsequently regulated by the interaction between IL-2 and the IL-2 receptor.
A T cell-specific protein tyrosine kinase, Lymphocyte cell kinase (hereinafter, referred to as "Lck") is a key molecule of TCR-mediated signaling and plays an important role in both T cell maturation and activation. The essential role of Lck in the various T cell function is accomplished by the interaction of SH2 and/or SH3 domain with other signaling effector molecules as well as catalytic activity of kinase domain.
SH2 domain, a conserved region in some regulatory proteins, comprises about 100 amino acids and plays a key role in the intracellular communication from a protein to another protein. Many of proteins with SH2 domain have been found to be involved in intracellular signal transduction. The binding specificity between SH2 domain of a protein and its ligand protein is determined by the sequences and structures of phosphotyrosine and nearby amino acids surrounding the binding site (Pawson, Nature, 373: 573-580, 1995).
The SH2 domain in Lck is one of the regulatory motifs of Lck, as is SH3 domain. When Lck is in inactive state, SH2 domain binds to intramolecular phosphotyrosine (Y505). Once Lck is activated, SH2 domain is released from autoregnlative mechanism, that is, the binding site of SH2 domain is exposed to the specific ligand proteins in T cells, allowing them to bind (Peri et al., Oncogene Res., 8: 2765-2772, 1993).
Several previous reports have tested on the key roles of Lck SH2 domain in T cell activation. One of the prominent studies exploited an Lck mutant, in which phosphotyrosine of ligand protein failed to bind to SH2 domain. In this Lck mutant, the signal transduction systems in T cells were found to be disordered, such as the T cell activation-induced calcium signaling and the signaling for the biosynthesis and secretion of IL-2, which are essential to the T cell proliferation (Xu and Littman, Cel, 74: 633-643, 1993; Straus et al., J. Biol. Chem., 271: 9976-9981, 1996; Lewis et al., J. Immunol., 159: 2292-2300, 1997). In summary, the SH2 domain of Lck shows the indispensability for efficient .alpha. chain phosphorylation, activation-induced intracellular Ca.sup.2+ mobilization and IL-2 gene expression in T cells.
Despite of the importance of SH2 domain in cellular signaling, little has been disclosed about the individual SH2 domain function. Study of the roles of tyrosine kinases like Lck in T cell receptor function has been hampered by lack of specific pharmacological inhibitors. Although several kinds of small molecule tyrosine kinase inhibitors such as herbinlycin and PP1/PP2 have been developed, but these drugs inhibit the kinase activity of tyrosine kinase and moreover, lack the specificity.